Parenchymal hepatocyte growth factors

ABSTRACT

A novel parenchymal hepatocyte growth factor originating in a human or animal liver, having an estimated molecular weight according to nonreductive SDS-PAGE of about 63,000 to about 69,000, an estimated molecular weight according to reductive SDS-PAGE of about 32,000 to about 36,000 and an estimated molecular weight according to gel filtration of about 60 to about 70 Kd; and having an activity of effecting the growth of parenchymal hepatocyte has been obtained from the hemihepatectomized tissue. Furthermore, a gene coding for the above substance has been obtained from the mRNA of the above tissue and it has thus become possible to mass-produce the above substance.

TECHNICAL FIELD

The present invention relates to a parenchymal hepatocyte growthsubstance originating in a human or animal liver and having an activityof effecting the extracellular growth of parenchymal hepatocytes, and agene coding for the above substance.

More particularly, the present invention relates to a parenchymalhepatocyte growth substance originating in a human or animal liver whichis expected to be effective for the treatment of liver diseases throughthe improvement of liver functions by proliferating normal hepatocytesin many liver diseases including hepatitis, liver cirrhosis andhepatoma, as well as a gene coding for the said substance.

BACKGROUND ART

It has been reported from old that when the liver suffers from variousdamages, regeneration of the liver occurs. The first report that partialhepatectomy of rat liver caused liver regeneration was made by Higgins,G. M., et al. in Arch. Pathol., 12, 186-202 (1931).

A hepatocyte growth factor (HGF) from rat platelets was found as asubstance for promoting the growth of liver cells by Toshikazu Nakamuraet al., see Biochem. Biophys. Res. Commun., 122, 1450-1459 (1984).

Likewise human HGF (hHGF) originating in human blood was discovered byAida et al., see Japanese Patent Application KOKAI No. 63-22526.

However, recent studies reveal that these substances (factors) are oneof cytokines produced in many organs (lung, liver, kidney, spleen, etc.)of rat and human and also exhibit the activities of acceleratinginfiltration of human cancer cells (renal tubule MDCK, lung cancer cells549) and stimulating the growth of other normal cells (epithelial cells,renal tubular cells, keratinocytes, melanocytes), see Nippon Rinsho, 50,1918 (1992).

A substance having diverse activities is anticipated to cause sideeffects in vivo and hence, it is desired to develop a substance capableof specifically stimulating the growth of hepatocytes.

In general, most of such substances (factors) are proteinaceous factorsproduced in a trace amount in vivo. In order to utilize such substancesas reagents for studies, agents for diagnosis and therapeutic agents, itis thus required to produce these substances in a large scale and alsofor this reason, it is desired to clarify the structure of suchsubstances.

DISCLOSURE OF INVENTION

The present inventors have made extensive studies with an attempt toobtain a novel gene coding for a hepatocyte growth factor from a ratliver and have succeeded in acquiring the novel gene coding for ahepatocyte growth factor from the rat residual liver after partialhepatectomy. Expression of this gene has resulted in successfullyobtaining a novel parenchymal hepatocyte growth substance andsuccessfully isolating and purifying the same substance from the ratresidual liver after partial hepatectomy. Furthermore, the same gene hasbeen successfully acquired from human liver. The present invention hasthus been accomplished. That is, as a first aspect the present inventionrelates to:

a parenchymal hepatocyte growth substance which is a proteinaceoussubstance originating in a human or animal liver and having thefollowing physico-chemical properties and physiological activities:

(1) an estimated molecular weight according to nonreductive SDS-PAGE ofabout 63,000 to about 69,000, an estimated molecular weight according toreductive SDS-PAGE of about 32,000 to about 36,000 and an estimatedmolecular weight according to gel filtration of about 60 to about 70 Kd;

(2) an activity of effecting the growth of parenchymal hepatocytes;

(3) said activity of effecting the growth of parenchymal hepatocytesbeing lost by a heat treatment at 95° C. for 5 minutes, a treatment withtrypsin or a treatment with chymotrypsin;

(4) adsorption to an ion exchange resin; and,

(5) non-adsorption to heparin.

As a second aspect, the present invention relates to a gene coding forthe parenchymal hepatocyte growth substance described above.

As a third aspect, the present invention relates to a process forproducing a parenchymal hepatocyte growth substance which comprisestransforming a host cell with an expression vector bearing the genecoding for the said parenchymal hepatocyte growth substance, culturingthe resulting transformant and collecting the hepatic parenchymal cellgrowth substance from the culture medium.

As a fourth aspect, the present invention relates to as process forproducing the parenchymal hepatocyte growth substance which comprisesisolating the parenchymal hepatocyte growth substance from thehomogenate of human or animal liver after partial hepatectomy, using anantibody to a partial peptide of the said parenchymal hepatocyte growthsubstance.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an amino acid sequence of the parenchymal hepatocyte growthsubstance originating in a human and animal liver and signal peptide(SEQ ID NOS:2 and 4).

FIG. 2 shows a nucleotide sequence of a gene coding for the parenchymalhepatocyte growth substance originating in a human and animal liver andsignal peptide, namely, the former half of cDNA of the parenchymalhepatocyte growth substance (SEQ ID NOS:1 and 3).

FIG. 3 shows a nucleotide sequence of a gene coding for the parenchymalhepatocyte growth substance originating in a human and animal liver andsignal peptide, namely, the latter half of CDNA of the parenchymalhepatocyte growth substance (SEQ ID NOS:1 and 3).

FIG. 4 shows a profile obtained by purification of the parenchymalhepatocyte growth substance originating in a rat liver through aSuperose 12 column.

FIG. 5 shows the results obtained by evaluation of the growth activityof the rat liver-originating parenchymal hepatocyte growth substance forrat parenchymal hepatocyte in terms of incorporation of ³ H-thymidineinto DNA.

FIG. 6 shows the results of electrophoretic analysis of the parenchymalhepatocyte growth substance originating in rat and human livers.

FIG. 7 shows the results obtained by evaluation of the growth activityof the human liver-originating parenchymal hepatocyte growth substancefor rabbit parenchymal hepatocytes in terms of incorporation of ³H-thymidine into DNA.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter the present invention will be described in detail withrespect to the steps of producing the parenchymal hepatocyte growthsubstance of the present invention and the gene coding for thesubstance, especially from rat and human livers.

Firstly, cDNA corresponding to the gene coding for the parenchymalhepatocyte growth substance that is considered to be specificallyproduced when rat liver is partially hepatectomized is surveyed andprepared by the following method.

(1) Construction of Liver cDNA Library of the Rat Residual Liver afterPartial Hepatectomy

It was first established by Higgins, G. M., et al that after partialhepatectomy, the rat liver is regenerated, see Arch. Pathol., 12, 186(1981)/

After two-third of the liver is removed for partial hepatectomy, theperitoneum and skin are sutured. Again the rat is lapatectomized 12hours after the liver removal when RNA synthesis increases and theresidual liver is all removed. RNA can be extracted according to theguanidine hydrochloride method by Deeley, R. G., et al., J. Biol. Chem.,252, 8310 (1980).

Furthermore, the RNA can be subjected to oligo (Dt) cellulose columnchromatography by a modification of the method by Amara, S. G., et al.,J. Biol. Chem., 255, 2645 (1980), to prepare mRNA.

cDNA library can be constructed by synthesizing double-stranded cDNAfrom this MRNA and incorporating the double-stranded cDNA into, e.g., E.coli plasmid or phage.

It is assumed that this cDNA library would contain cDNA corresponding tothe gene coding for the parenchymal hepatocyte growth substance expectedto be specifically produced upon partial hepatectomy of a rat liver.

(2) Preparation of cDNA Probe in the Rat Residual Liver after partialHepatectomy and cDNA Probe of Sham-Operated Rat Liver

a) Preparation of cDNA probe in the rat residual liver after partialhepatectomy

A part of the MRNA prepared by the oligo (Dt) cellulose columnchromatography is used to synthesize double-stranded cDNA. Herein cDNAis isotope-labelled with α-³² P! dCTP according to the nick translationmethod, which is made cDNA probe of the rat residual liver after partialhepatectomy.

b) Preparation of cDNA Probe of Sham-Operated Rat Liver

After a rat is lapatectomized but no liver is removed, the peritoneumand skin are sutured. Again the rat is lapatectomized 12 hours after theoperation and the liver is all removed. RNA is extracted according tothe guanidine hydrochloride method and subjected to oligo (Dt) cellulosecolumn chromatography to prepare mRNA. Using the mRNA as a template,double-stranded cDNA is synthesized while labelling with α-³² P! DCTPaccording to the nick translation method. The double-stranded cDNA ismade CDNA probe of the sham-operated rat liver.

(3) Transfer of cDNA Library Clones onto a Nitrocellulose Membrane

Two nitrocellulose membrane sheets sterilized are previously put on anagar medium plate and all of the cDNA library clones obtained in (1) aretransferred thereon by every two sheets under the same conditions. Theclone-transferred nitrocellulose membranes are treated with an alkaliand immobilized at 80° C.

(4) Hybridization by the cDNA Probe of the Residual Rat Liver afterPartial Hepatectomy and by the CDNA Probe of Sham-Operated Rat Liver

With respect to one membrane out of the two immobilized membranes ofeach clone in (3) above, hybridization is performed using the cDNA probeof the residual rat liver after partial hepatectomy, prepared in (2) a).For another membrane, hybridization is performed using the cDNA probe ofsham-operated rat liver prepared in (2) b) above.

These membranes are overlaid on an X ray film, respectively andsensitized in an X ray film cassette for autoradiography. The clonessensitized with the cDNA probe from the rat residual liver after partialhepatectomy and those sensitized with the cDNA probe from thesham-operated rat liver are compared with the same clones to select onlythe clones that are more strongly sensitized with the cDNA probe fromthe residual liver than with the cDNA probe from the sham-operatedliver. The thus obtained clones appear to contain cDNA clone coding forthe parenchymal hepatocyte growth substance expected to be specificallyproduced by hepatectomy.

(5) Selection by Hybridization

Each of the selected clonal cDNAs is amplified by, e.g., PCR and theamplified cDNAS are immobilized or fixed onto a nitrocellulose membrane,respectively.

On the other hand, mRNAs are prepared from the rat residual liver afterpartial hepatectomy in a manner similar to (1) described above toprepare a mRNA solution having a high nucleotide concentration. Theclonal CDNA-immobilized nitrocellulose membrane described above isimmersed in the solution as a probe.

By this procedure, mRNA complementary to the clonal cDNA fixed onto eachnitrocellulose membrane is adsorbed to the membrane. Thereafter themembrane is immersed in a solution having a low nucleotide concentrationto obtain complementary MRNA.

(6) Expression of Protein

The MRNA solution obtained by hybridization selection is injected in atrace amount into Xenopus laevis oocyte. Incubation at 20° C. for 24 to48 hours in a Barths' medium causes translation of the mRNA injected toexpress a protein. Where this protein is secretory, the protein can beproduced in the medium.

(7) Evaluation of Hepatocyte Growth Activity

The hepatocyte growth activity of the protein expressed in (6) above isevaluated by the following method.

The parenchymal hepatocytes are separated from the rat liver to performincubation of the primary liver cells. Upon the incubation, the culturebroth containing the expressed protein obtained in (6) above is addedthereto. In addition, ³ H-thymidine is added to the medium and the ³ Hincorporated into the parenchymal hepatocytes after a definite period oftime is counted with a liquid scintillation counter. By the change of ³H incorporated into the parenchymal hepatocytes, the hepatocyte growthactivity can be evaluated.

mRNA capable of expressing the protein having a hepatocyte growthactivity is selected and using the corresponding clonal cDNA, thefollowing nucleotide sequence is analyzed so that the nucleotidesequence of the gene coding for the parenchymal hepatocyte growthsubstance can be determined.

(8) Analysis of Nucleotide sequence of the Gene cDNA Coding for theParenchymal Hepatocyte Growth Substance

In order to determine the nucleotide sequence of the gene correspondingto the mRNA capable of secreting the substance having a hepatocytegrowth activity obtained in (7) described above, sequencing according tothe dideoxy method or the like may be performed. The nucleotide sequenceof the gene coding for the parenchymal hepatocyte growth substance isdetermined and based on the sequence determined, the amino acid sequencecorresponding to the nucleotide sequence, namely, the amino acidsequence of the parenchymal hepatocyte growth substance can bespeculated.

(9) Preparation of Anti-Partial Peptide Fragment Antibody of theParenchymal Hepatocyte Growth Substance and Preparation of theParenchymal Hepatocyte Growth Substance

Based on the nucleotide sequence of the gene coding for the parenchymalhepatocyte growth substance, the corresponding amino acid sequence canbe determined. Among them, a peptide corresponding to about 20 optionalamino acid residues is synthesized and an animal such as rabbit isimmunized with the peptide to prepare an antibody specific to theparenchymal hepatocyte growth substance. The antibody is bound to acolumn. Using the column, the parenchymal hepatocyte growth substance ofthe present invention can be isolated and extracted from the ratresidual liver homogenate after partial hepatectomy.

(10) Preparation of the Parenchymal hepatocyte Growth Substance by GeneExpression

The parenchymal hepatocyte growth substance-encoding CDNA obtained in(7) described above is transduced in an appropriate plasmid, e.g., pSVL(made by Pharmacia). After transforming, e.g., COS-7 cells, with thisexpression vector, the resulting transformant is cultured to express theparenchymal hepatocyte growth substance. The parenchymal hepatocytegrowth substance of the present invention can be collected from theculture medium.

The gene coding for the parenchymal hepatocyte growth substanceoriginating in a rat liver can be prepared by the steps described aboveand its nucleotide sequence can be determined. Furthermore, theparenchymal hepatocyte growth substance can be obtained by expressing ofthe gene. Alternatively, a partial peptide of the parenchymal hepatocytegrowth substance is synthesized and by using an antibody to the peptide,the parenchymal hepatocyte growth substance can be isolated from the ratresidual liver homogenate after partial hepatectomy.

Various mRNAS of animal cells or animal tissues such as human liver mRNAare commercially available and can be purchased from Clontech Co., Ltd.,etc. to utilize them for the present invention.

Accordingly, cDNA library can be prepared by purchasing, e.g., humanliver mRNA, synthesizing CDNA using the mRNA and incorporating this cDNAinto a plasmid or phage.

From the resulting cDNA library cDNA coding for human liver-derivedparenchymal hepatocyte growth substance can be acquired using as a probethe full length of or a part of cDNA coding for the parenchymalhepatocyte growth substance, which is obtained from the rat liverdescribed above. Then, the nucleotide sequence of this cDNA can bedetermined according to the dideoxy method. The amino acid sequence canbe deduced from the nucleotide sequence and an antibody can be preparedusing as an antigen a peptide containing a part of the amino acidsequence; using the antibody, the parenchymal hepatocyte growthsubstance derived from human liver can be isolated and purified in amanner similar to above.

Alternatively, human liver cDNA can be transduced into, e.g., plasmidPSVL in a manner similar to above thereby to construct an expressionvector and transform, e.g., COS-7 cells with the expression vector.Then, the resulting transformant is cultured and the human liver-derivedparenchymal hepatocyte growth substance can also be obtained from theculture medium.

The thus obtained parenchymal hepatocyte growth substance of the presentinvention originating from a human or animal liver has the followingproperties.

(1) Molecular Weight

An estimated molecular weight according to nonreductive SDS-PAGE isabout 63,000 to about 69,000, an estimated molecular weight according toreductive SDS-PAGE is about 32,000 to about 36,000 and an estimatedmolecular weight according to gel filtration is about 60 to about 70 Kd.

(2) Physiological Activity

The substance has an activity of effecting the growth of parenchymalhepatocytes.

(3) The activity of effecting the growth of parenchymal hepatocyte islost by a heat treatment at 95° C. for 5 minutes, a treatment withtrypsin or a treatment with chymotrypsin.

(4) The substance is adsorbed to an ion exchange resin such as DEAESepharose or Q-Sepharose.

(5) The substance is not adsorbed to heparin.

In addition, the parenchymal hepatocyte growth substance has thefollowing characteristics.

The human liver-derived hepatic parenchymal cell growth substance has asits N-terminal amino acid sequence an amino acid sequence represented byformula (1) below (SEQ ID NO:5):

    (N-terminus) Leu Glu Asp Cys Ala Gln Glu Gln Met Arg Leu Arg Ala Gln Val Arg Leu Leu Glu Thr Arg Val Lys Gln Gln Gln Val Lys Ile Lys (C-terminus)

wherein Leu, Glu, Asp, Cys, Ala, Gln, Met, Arg, Val, Thr, Lys and Ile

wherein L, E, D, C, A, Q, M, R, V, L, T, K and I represent leucine,glutamic acid, aspartic acid, cysteine, alanine, glutamine, methionine,arginine, valine, leucine, threonine, lysine and isoleucine,respectively.

The full-length amino acid sequence of the human liver-derivedparenchymal hepatocyte growth substance deduced from its CDNA nucleotidesequence appears to correspond to the amino acid sequence from 23leucine to 312 isoleucine in the amino acid sequence shown in FIG. 1,lower column.

The rat liver-derived parenchymal hepatocyte growth substance has as itsN-terminal amino acid sequence an amino acid sequence represented byformula (2) below (SEQ ID NO:6):

    (N-terminus) Asp Glu Asn Cys Leu Gln Glu Gln Val Arg Leu Arg Ala Gln Val Arg Gln Leu Glu Thr Arg Val Lys Gln Gln Gln Val Val Ile Ala (C-terminus)

wherein Asp, Glu, cys, Leu, Gln, Val and Ile have the same significanceas defined above and N represents asparagine.

The full-length amino acid sequence of the rat liver-derived parenchymalhepatocyte growth substance deduced from its cDNA nucleotide sequenceappears to correspond to the amino acid sequence from 25 aspartic acidto 314 valine in the amino acid sequence shown in FIG. 1, upper column(SEQ ID NO:2).

An example of the gene coding for the aforesaid entire amino acidsequence of the human liver-derived parenchymal hepatocyte growthsubstance includes a gene having nucleotide sequence from codon CTCstarting from 117 to codon ATT starting from 984 (corresponding to theportion shown by solid line with arrow in FIGS. 2 and 3) in thenucleotide sequences shown in FIGS. 2 and 3, lower column.

An example of the gene coding for the aforesaid entire amino acidsequence of the rat liver-derived parenchymal hepatocyte growthsubstance includes a gene having nucleotide sequence from codon GATstarting from 122 to codon GTT starting from 990 (corresponding to theportion shown by solid line with arrow in FIGS. 2 and 3) in thenucleotide sequences shown in FIGS. 2 and 3, upper column (SEQ IDNO:3-4).

These genes are determined according to the procedures for analysis ofcDNA nucleotide sequence at step (8) in the process described above.Using these genes, the parenchymal hepatocyte growth substance of thepresent invention can be prepared according to the procedures shown instep (10) described above.

Hereinafter the present invention is described below in more detail byreferring to Examples.

EXAMPLE 1

The following is an example for preparing the rat liver-derivedparenchymal hepatocyte growth substance and the gene coding for thesubstance.

(1) Construction of Liver cDNA Library of the Rat Residual Liver afterPartial Hepatectomy

Sprague-Dawley male rat of 8 weeks age was lapatectomized in about 20-25mm length from the xiphoid process along the center to withdraw theliver and cut off the right lateral lobe, left lateral lobe and the leftmedian lobe. That is, about two-third of the liver was removed.Immediately thereafter, the peritoneum and skin were sutured. The ratwas again lapatectomized 12 hours after the liver removal when RNAsynthesis increased and the residual liver was all removed. To the liverremoved was added 10-fold volume of 8M guanidine hydrochloride/20 Mmsodium acetate buffer (Ph 5.5). After thoroughly mixing with ahomogenizer, the mixture was centrifuged at 18,000×g to obtain thesupernatant as the sample solution. 5.7M cesium chloride was previouslycharged in a tube for ultracentrifugation and 8M guanidinehydrochloride/20 Mm sodium acetate (Ph 5.5) was overlaid thereon and,the sample solution was further overlaid thereon. Centrifugation wasperformed at 20° C. and 25 rpm for 20 hours.

By the above procedure, RNA was isolated as pellets. This RNA wasdissolved in a suitable quantity of sterile water. An equal quantity of0.4 Mm NaCl/20 Mm Tris hydrochloride buffer (Ph 7.4) was added to thesolution and the mixture was poured onto an oligo (Dt) cellulose columnwhich had been previously equilibrated with 0.2M NaCl/10 Mm Trishydrochloride buffer (Ph 7.4). After rinsing with the same solution,elution was effected with 10 Mm Tris hydrochloride buffer/1 Mm EDTA(ethylenediamine tetraacetate) (Ph 7.4). The eluate was fractionated.With respect to a part of the fraction, absorbance was measured at 260nm using a spectrophotometer to collect the elution peak. An equalquantity of 4M ammonium acetate was added to the elution peak collectedfollowed by stirring. A 2-fold volume of ethanol was added to thestirred solution followed by thorough stirring. After allowing to standat -20° C. for 3 hours, centrifugation was carried out at 4° C. for 5minutes at 15,000 rpm and the resulting precipitates were mRNA to beused for the following experiment. According to the method of Land, H.(Nucl. Acids. Res., 9, 2251 (1981)), double-stranded CDNA wassynthesized from the mRNA.

Next, double-stranded cDNA synthesized by the method of Maniatis, T.(Molecular Cloning, 249 (1982)) was inserted into E. coli plasmid pUC8.This recombinant DNA was transfected to E. coli HB101 fortransformation. Approximately 68,000 clones were obtained as suchtransformed clones, which were made cDNA library of the rat residualliver after partial hepatectomy.

(2) Selection of Clone specifically expressed after partial Hepatectomy

The transformed clones in the cDNA library of the rat residual liverafter partial hepatectomy were transferred onto two filters,respectively. The transfer onto the filters was conducted by overlayingthe filters previously sterilized onto ampicillin-supplemented (50μg/ml) LB agar medium (1% bacto-trypton, 0.5% yeast extract, 1% NaCl, pH7.5) and transferring the transformed clones onto the two filters with abamboo spit or tooth pick similarly sterilized. Incubation at 37° C.overnight resulted in proliferation of the E. coli clones on the filtersto form colonies. The filters were treated with an alkali by amodification of the Grunstein, M. et al. method (Proc. Natl. Acad. Sci.USA, 72, 3691 (1975)) to fix the colonies onto the filters.

Using as a probe α-³² p! dCTP-labeled cDNA of the rat residual liverafter partial hepatectomy, hybridization was performed on one of the twofilters fixed and on another filter, hybridization was performed usingas a probe α-³² p! dCTP-labeled cDNA of the sham-operated rat liver. Theα- ³² p! dCTP-labeled cDNA of the rat residual liver after partialhepatectomy which was employed hereinabove was obtained by synthesizingdouble-stranded cDNA using a part of the mRNA obtained by oligo (dT)cellulose column chromatography in step (1) described above andlabelling the cDNA with radioactive α-³² p! dCTP according to the nicktranslation method. The α-³² p! dCTP-labeled cDNA of the sham-operatedrat liver was obtained by lapatectomizing a rat, suturing the peritoneumand the skin without removing the liver, again lapatectomizing the rat12 hours after the operation to remove all of the residual liver,preparing mRNA in a manner similar to step (1) described above, andlabelling the cDNA with radioactive α-³² P! dCTP according to the nicktranslation method.

After the hybridization, each filter was washed at room temperature with2×SSC (0.3M NaCl, 30 mM sodium citrate)/0.1% SDS (sodium dodecylsulphate) and further with 0.1×SSC/0.1% SDS at 50° C. followed bydrying.

The dried filters were overlaid on an X ray film, respectively, whichwas sensitized at -80° C. in an X ray film cassette for autoradiography.The clones sensitized with the cDNA probe from the rat residual liverafter partial hepatectomy and those sensitized with the cDNA probe fromthe sham-operated rat liver were compared with the same clones to selectonly 40 clones that were more strongly sensitized with the cDNA probefrom the residual liver than with the cDNA probe from the sham-operatedliver.

(3) Evaluation of Hepatocyte Growth Activity

The aforesaid 40 clones cDNAs that appear to be specifically producedand amplified in the liver tissue in association with liver regenerationwere amplified and adsorbed onto nitrocellulose filters, respectively,according to the method of Taniguchi et al. (Proc. Natl. Acad. Sci. USA,77, 4003 (1980)).

The mRNA taken out of the residual liver 12 hours after partialhepatectomy was further adsorbed onto these filters in the presence ofNaCl. After the filters were washed with NaCl, elution was performedwith water to selectively obtain mRNA having a sequence complementary tothe previously adsorbed clone cDNA. According to the method described inColman, A., Transcription and translation, 271 (1984), this mRNA wascondensed and injected in a trace amount into Xenopus laevis oocytefollowed by incubation at 20° C. in a modified Barths' medium. After 48hours, the culture medium was recovered and the growth activity of theparenchymal hepatocytes was evaluated as follows.

By circulation through the rat liver, the parenchymal hepatocytes wereseparated and cultured to provide for the evaluation. That is, Williams'E medium supplemented with 0.05% collagenase is circulated through theliver of Wistar strain male rat of 8 weeks age to separate hepatocytes.The separated cells are centrifuged at 50 g x for a minute and thesupernatant was removed. The precipitated cells are gently suspended inWilliams' E medium and the suspension was centrifuged several times in asimilar manner. By this procedure, parenchymal hepatocytes can beobtained in a high purity. The cells are inoculated in a density of2×10⁴ /cm² on a Petri dish previously coated with a collagen solutionuniformly and then dried. Incubation is carried out at 37° C. in thepresence of 5% carbon dioxide gas. The medium is exchanged with a freshmedium 3 to 6 hours after. At the same time, a specimen (the aforesaidculture medium obtained by culturing Xenopus laevis oocyte injected withmRNA) and is added to the medium in a definite amount. Twenty hoursafter, 0.1 μCi of ³ H-thymidine is added to the medium followed byincubation for further 6 to 20 hours. Then the ³ H incorporated into thecells is counted. That is, the medium is removed and the cells aregently rinsed several times with PBS (phosphate buffer/physiologicalsaline). The cells are further treated at 50° C. in the presence of 2NNaOH. The treated cells are stripped out of the Petri dish anddissolved. After neutralization, the amount of ³ H is counted with aliquid scintillation counter. Where a substance having the growthactivity is present in the specimen, the amount of ³ H incorporated intothe hepatocytes increases. For evaluation of the hepatocyte growthactivity in such a manner, the parenchymal hepatocytes can be separatednot only from the rat liver but also the liver of animals such as mouse,rabbit, etc. and provided for the evaluation.

According to the above method, the growth activity of the parenchymalhepatocytes was evaluated. Among 40 culture media (corresponding to the40 clones) obtained by injecting the complementary mRNA, obtained instep (3) above using as templates the 40 clones cDNA obtained in step(2) above, into Xenopus laevis oocyte and culturing the oocyte, a highlypotent parenchymal hepatocyte growth activity was noted in one medium(clone).

For control, comparison was made using the Xenopus laevis oocyte culturemedium injected only with distilled water in a trace amount. The proteinexpressed only in the full-length mRNA-injected Xenopus laevis oocyteculture medium showed a molecular weight of about 60,000 to about 70,000according to electro-phoresis (nonreductive SDS-PAGE).

(4) Determination of Nucleotide Sequence of the Gene cDNA Coding for theParenchymal Hepatocyte Growth Substance

The nucleotide sequence of clonal cDNA coding for the gene productshowing a parenchymal hepatocyte growth activity, which was selected instep (3) above, was determined by the method of Nakamura et al. (SaiboKogaku, 7, 712 (1988)). This clonal cDNA was considered to be deleted ofthe 5' end so that the following procedure was conducted to determinethe site at the 5' end.

In order to newly prepare cDNA bearing the 5' end from the rat livermRNA, firstly the residual liver was removed from the rat 12 hours afterpartial hepatectomy, in a manner similar to step (1) above, thereby toobtain mRNA. The mRNA obtained from the hemihepatectomized tissue washeated at 70° C. for 10 minutes and then quenched.

The oligonucleotide of 40 nucleotides complementary to the 40nucleotides of the clonal cDNA at the 5' end obtained in step (3) above,which corresponds to the nucleotide sequence surrounded by square inFIG. 2 (SEQ ID NO:9):

    (5' end) TGCCGTCAGATCGTCTC TGAATTACAGTCCATCCTCCTCC (3' end)

was synthesized. Using this oligonucleotide as a primer and theaforesaid mRNA obtained from the hemi-hepatectomized tissue as atemplate, cDNA at the 5' end site was prepared. With respect to the thusobtained cDNA, the nucleotide sequence of the cDNA at the 5' end sitewhich encodes the gene product was determined according to the method ofNakamura et al. (Saibo Kogaku, 7, 712 (1988)).

By the above procedure, the nucleotide sequence of the full-length cDNAcoding for the rat liver-derived substance showing the parenchymalhepatocyte growth activity was revealed. The nucleotide sequence isshown in FIGS. 2 and 3, upper column (SEQ ID NO:1). The amino acidsequence of the rat liver-derived parenchymal hepatocyte growthsubstance shown in FIG. 1, upper column was deduced from the nucleotidesequence of FIGS. 2 and 3 (SEQ ID NO:2).

(5) Acquirement of Antibody to the Partial Peptide deduced from theNucleotide Sequence and preparation of an Antibody Column

Based on the nucleotide sequence of the gene determined in step (4)above, a peptide of hydrophilic portion was synthesized.

Three peptides of hydrophilic portion were prepared from the amino acidsequence shown in FIG. 1, upper column, which was deduced from thenucleotide sequence obtained in step (4) above. The amino acid sequencesfrom 125 to 143, from 180 to 201 and from 236 to 256 were designatedPeptides 1, 2 and 3, respectively. Each of three Japanese albino rabbitswas boostered with Peptides 1 to 3, respectively, in a dose of 2 mg, onea week for 5 weeks.

As the result, antiserum obtained from Peptide 2 maintained the antibodytiter even though it was diluted to 50,000-fold. From this serum theantibody was purified through a protein G column.

The purified antibody was bound to CNBr-activated Sepharose 4B toprepare an antibody column (Rαpep 200 IgG-Sepharose 4B column).

(6) Purification of the Parenchymal Hepatocyte Growth Substance

After the rat residual liver 24 hours after 70% partial hepatectomy wasremoved, the liver was thoroughly homogenized with a homogenizer.Centrifugation at 100,000×g at 4° C. for 30 minutes followed a treatmentwith heparin-cellurofine column. The non-adsorbed fraction was adsorbedto the antibody column prepared in step (5) above followed by elutionwith 0.15M ammonia -0.15M NaCl. The eluted fraction was concentrated.Gel filtration of the concentrate through a Superose 12 column gave aprotein fraction reactive with the antibody at a molecular weight ofabout 65 kd. The purification profile through the Superose 12 column isshown in FIG. 4. With regard to the protein fraction thus obtained, theactivity was determined according to the evaluation of hepaticparenchymal cell growth activity described in detail in step (3) above.The activity shown in FIG. 5 was appreciated.

Analysis of the amino acid sequence of the N-terminus of the purifiedparenchymal hepatocyte growth substance by Sequencing Analyzer (AppliedBiosystems, Model 473A) revealed that the substance had the amino acidsequence shown by formula (2) below (SEQ ID NO:6).

    (N-terminus) Asp Glu Asn cys Leu Gln Glu Gln Val Arg Leu Arg Ala Gln Val Arg Gln Leu Glu Thr Arg Val Lys Gln Gln Gln Val Val Ile Ala (C-terminus)

(7) Expression of Gene

In order to insert the gene coding for the parenchymal hepatocyte growthsubstance into COS-7 cells (Rikagaku Kenkyusho) originating from thekidney of African green monkey to produce a protein, the full-lengthcDNA coding for the parenchymal hepatocyte growth substance obtained instep (4) above, namely, cDNA having the nucleotide sequence at the uppercolumn of the portion shown by dotted line with arrow in FIGS. 2 and 3,was inserted into plasmid pSVL (made by Pharmacia) bearing SV40 latepromoter gene. Firstly this recombinant DNA was transfected to E. coliHB101 to perform DNA amplification. This recombinant DNA is made pSVLR.The thus obtained E. coli HB101 transfected with pSVLR was namedEscherichia coli HB101-pR, which was deposited in the Life ScienceResearch Institute of the Agency of Industrial Science and Technology ofJapan on Nov. 17, 1992 and accepted under FERN P-13289. Then the strainwas transferred to the international deposit and given an accessionnumber of FERM BP-4594 on Mar. 3, 1994, pursuant to the Budapest Treaty.

The DNA was further transfected to COS-7 followed by incubation. DMEMwas used as a medium and incubation was conducted at 37° C. for 4 to 6days in the presence of 5% CO₂. The culture medium was recovered andsubjected to electrophoresis by SDS-PAGE to obtain a band showing amolecular weight of about 63,000 to about 69,000 under nonreductiveconditions and a molecular weight of about 32,000 to about 36,000 underreductive conditions. This was a band which was not present in thecontrol (culture medium of the cells transfected only with pSVL). FIG. 6indicates the results of the electrophoresis.

The culture medium described above was added to the primary culturemedium of the hepatocytes and the hepatocyte growth activity wasexamined according to the method for determining the parenchymalhepatocyte growth activity shown in step (3) above. It was confirmedthat the hepatocyte growth activity of the primary culture was present.No growth activity was observed in the culture medium of the cellstransfected only with pSVL. It is thus assumed that this gene productwould be the active substance.

The same gene as described above was inserted into vector-pCDL-SRα296bearing HTLV-1.LTR gene, which was then transfected to Verots S-3(Rikagaku Kenkyusho) originating from the kidney of African greenmonkey. Incubation was carried out in a similar manner. The culturemedium was recovered and subjected to electro-phoresis by SDS-PAGE togive a band showing a molecular weight of about 63,000 to about 69,000under nonreductive conditions and a molecular weight of about 32,000 toabout 36,000 under reductive conditions. The culture medium describedabove was added to the primary culture medium of the hepatic cells andthe hepatocyte growth activity was examined. It was confirmed that thehepatocyte growth activity of the primary culture was present.

Based on the findings on the molecular weight, the amino acid sequenceshown in FIG. 1 and the amino acid sequence of the N-terminal portiondescribed in step (6) above, it is considered that in the amino acidsequence shown in FIG. 1, upper column, the rat liver-derivedparenchymal hepatocyte growth substance has the amino acid sequence from25 aspartic acid to 314 valine (portion shown by the solid line witharrow in FIG. 1 (SEQ ID NO:2 ), takes a cyclic structure through twodisulfide bonds (S--S bonds) within the molecule due to 5 cysteineresidues contained in the amino acid sequence, and forms a homodimer inwhich two of such a molecule are bound to each other through the S--Sbonds.

The protein expressed and recovered in step (7) above appears to be aprotein in which signal peptide (amino acid sequence at the underlinedportion in FIG. 1) corresponding to the amino acid sequence from 1methionine to 24 glycine shown in FIG. 1, upper column is excised toform a dimer.

The nucleotide sequence of the gene coding for the rat liver-derivedparenchymal hepatocyte growth substance is the nucleotide sequence(portion shown by the solid line with arrow in FIGS. 2 and 3) from codonGAT starting from 122 and to codon GTT starting from 990 in thenucleotide sequence shown in FIGS. 2 and 3, upper column (SEQ ID NO:1).

(8) Thermal Stability

The parenchymal hepatocyte growth substance obtained in steps (6) and(7) above was treated at 95° C. for 5 minutes. When the treated solutionwas added to the culture medium of the rat primary culture hepatocytes,no growth activity was exhibited on the hepatocytes.

(9) Stability against Trypsin and Chymotryosin

The parenchymal hepatocyte growth substance (5 μg/ml) obtained in steps(6) and (7) above was treated with trypsin (made by Sigma; 0.1 mg/ml) at37° C. for 30 minutes. After trypsin was removed, the treated matter wasadded to the culture medium of the rat primary culture hepatocytes butno growth activity was exhibited on the hepatocytes.

When the substance was treated with chymotrypsin (made by Sigma; 0.1mg/ml) at 37° C. for 30 minutes, the parenchymal hepatocyte growthactivity was also lost.

(10) Column Characteristic P 1. Heparin-Sepharose

The culture medium, 1 liter, containing the parenchymal hepatocytegrowth substance and produced by genetic recombination obtained in step(7) above was dialyzed to 20 mM phosphate buffer (pH 6.5)/0.5M NaCl for16 hours and the dialysate was thoroughly equilibrated. Then thedialysate was poured through a heparin-Sepharose column (2×20 cm)equilibrated with the same buffer. After thoroughly washing with thesame buffer, elution was performed with 20 mM phosphate buffer (pH6.5)/2.0M NaCl. The parenchymal hepatocyte growth substance was found inthe non-adsorbed fraction but absent in the eluted fraction. It wasconfirmed by electrophoresis (SDS-PAGE) and by the method fordetermining the parenchymal hepatocyte growth activity of the ratprimary culture as to whether or not the substance was present. Theresults reveal that the parenchymal hepatocyte growth substance was notadsorbed to heparin-Sepharose.

2. CM Sepharose

The culture medium, 1 liter, containing the parenchymal hepatocytegrowth substance and produced by genetic recombination obtained in step(7) above was dialyzed to 20 mM phosphate buffer (pH 6.5) for 16 hours.The dialysate was thoroughly equilibrated and then poured through a CMSepharose column (2×20 cm) equilibrated with the same buffer. Afterthoroughly washing with the same buffer, elution was performed with 20mM phosphate buffer (pH 6.5)/2.0M NaCl. The hepatic parenchymal cellgrowth substance was found in the non-adsorbed fraction but absent inthe eluted fraction. It was confirmed by electrophoresis (SDS-PAGE) andby the method for determining the parenchymal hepatocyte growth activityof the rat primary culture as to whether or not the substance waspresent. The results reveal that the parenchymal hepatocyte growthsubstance was not adsorbed to CM Sepharose.

3. DEAE cellulose

The culture medium, 1 liter, containing the parenchymal hepatocytegrowth substance and produced by genetic recombination obtained in step(7) above was dialyzed to 20 mM Tris hydrochloride buffer (pH 8.0) for16 hours. The dialysate was thoroughly equilibrated and then pouredthrough a DEAE Sepharose column (2×20 cm) equilibrated with the samebuffer. After thoroughly washing with the same buffer, gradient elutionwas performed with 20 mM Tris hydrochloride buffer (pH 8.0)/2.0M NaCl.The parenchymal hepatocyte growth substance was found in a small amountin the non-adsorbed fraction and also eluted in the fraction of 0.5-0.8MNaCl. It was confirmed by electrophoresis (SDS-PAGE) and by the methodfor determining the hepatic parenchymal cell growth activity of the ratprimary culture as to whether or not the substance was present. Theresults reveal that the parenchymal hepatocyte growth substance wasalmost all adsorbed to DEAE Sepharose.

EXAMPLE 2

The parenchymal hepatocyte growth substance originating in a human liveris described below with reference to the following example.

(1) Selection of Human Liver cDNA

In order to obtain from a human liver cDNA having a high homology tocDNA coding for the rat liver-derived parenchymal hepatocyte growthsubstance, cDNA was prepared using human liver mRNA (made by Toyobo :Clontec). The cDNA was packaged in λgt11phage to construct a library.

From this library, cDNA having a high homology to the rat gene wasobtained using as a probe cDNA of the rat liver-derived parenchymalhepatocyte growth substance obtained in Example 1, step (4), in whichthe 5' end site is deleted.

(2) Analysis of the Nucleotide Sequence of cDNA

In order to determine the nucleotide sequence of the gene cDNA obtainedin step (1) above, the nucleotide sequence was determined according tothe dideoxy method by a modification of the Nakamura et al. method(Saibo Kogaku, 7, 712 (1988)). The nucleotide sequence is shown in FIGS.2 and 3, lower column (SEQ ID NO:3). The amino acid sequence deducedfrom the nucleotide sequence is shown in FIG. 1, lower column (SEQ IDNO:4).

(3) Expression of Gene

In order to insert the gene coding for the human liver-derivedparenchymal hepatocyte growth substance into COS-7 cells (RikagakuKenkyusho) originating from the kidney of African green monkey toproduce a protein, the full-length cDNA coding for the parenchymalhepatocyte growth substance obtained in step (1) above, namely, cDNAhaving the nucleotide sequence at the lower column of the portion shownby the dotted line with arrow in FIGS. 2 and 3, was inserted intoplasmid pSVL (made by Pharmacia) bearing SV40 late promoter gene.Firstly this recombinant DNA was transfected to E. coli HB101 to performDNA amplification. This recombinant DNA is made pSVLH. The thus obtainedE. coli HB101 transfected with PSVLH was named Escherichia coliHB101-pH, which was deposited in the Life Science Research Institute ofthe Agency of Industrial Science and Technology of Japan on Nov. 17,1992 and accepted under FERM P-13288. Then the strain was transferred tothe international deposit and given an accession number of FERM BP-4593on Mar. 3, 1994, pursuant to the Budapest Treaty.

The DNA was further transfected to COS-7 followed by incubation. DMEMwas used as a medium and incubation was conducted at 37° C. for 4 to 6days in the presence of 5% CO₂. The culture medium was recovered andsubjected to electrophoresis by SDS-PAGE to obtain a band showing amolecular weight of about 63,000 to about 69,000 under nonreductiveconditions and a molecular weight of about 32,000 to about 36,000 underreductive conditions. This was a band which was not present in thecontrol (culture medium of the cells transfected only with PSVL). FIG. 6indicates the results of the electrophoresis.

The culture medium described above was added to the primary culturemedium of the hepatocytes and the hepatocyte growth activity wasexamined according to the method for determining the parenchymalhepatocyte growth activity shown in Example 1, step (3). It wasconfirmed that the hepatocyte growth activity of the primary culture waspresent. The results of the activity evaluation are shown in FIG. 7. Nogrowth activity was observed in the culture medium of the cellstransfected only with pSVL. It is thus assumed that this gene productexpressed would be the active substance.

The same gene as described above was inserted into vector-pCDL-SRα296bearing HTLV-1.LTR gene, which was then transfected to Verots S-3(Rikagaku Kenkyusho) originating from the kidney of African greenmonkey. Incubation was carried out in a similar manner. The culturemedium was recovered and subjected to electro-phoresis by SDS-PAGE togive a band showing a molecular weight of about 63,000 to about 69,000under nonreductive conditions and a molecular weight of about 32,000 toabout 36,000 under reductive conditions. The culture medium describedabove was added to the primary culture medium of the hepatic cells andthe hepatocyte growth activity was examined. It was confirmed that thehepatocyte growth activity of the primary culture was present.

The parenchymal hepatocyte growth substance was recovered and purifiedfrom the culture medium. Analysis of the amino acid sequence of theN-terminus of the purified substance by Sequencing Analyzer (AppliedBiosystems, Model 473A) revealed that the substance had the amino acidsequence shown by formula (1) below as the N-terminal amino acidsequence (SEQ ID NO:5).

    (N-terminus) Asp Glu Asn cys Leu Gln Glu Gln Val Arg Leu Arg Ala Gin Val Arg Gln Leu Glu Thr Arg Val Lys gln Gln Val Val Ile Ala (C-terminus)

Based on the foregoing findings, it is considered that in the amino acidsequence shown in FIG. 1, lower column, the human liver-derivedparenchymal hepatocyte growth substance has the amino acid sequence from23 leucine to 312 isoleucine (portion shown by the solid line with arrowin FIG. 1), takes a cyclic structure through two disulfide bonds withinthe molecule as in the rat liver-derived substance and forms a homodimerin which two of such a molecule are bound to each other through thedisulfide bonds. The protein obtained by expression of the gene appearsto be a protein in which signal peptide (amino acid sequence at theunderlined portion in FIG. 1) corresponding to the amino acid sequencefrom 1 methionine to 22 alanine shown in FIG. 1, lower column is excisedto form a dimer.

The nucleotide sequence of the gene coding for the human liver-derivedparenchymal hepatocyte growth substance is the nucleotide sequence(portion shown by the solid line with arrow in FIGS. 2 and 3) from codonCTC starting from 117 and to codon ATT starting from 984 in thenucleotide sequence shown in FIGS. 2 and 3.

(4) Thermal Stability

The parenchymal hepatocyte growth substance obtained in step (3) abovewas treated at 95° C. for 5 minutes. When the treated solution was addedto the culture medium of the rat primary culture hepatocytes, no growthactivity was exhibited on the hepatocytes.

(5) Stability against Trypsin and Chymotrypsin

The human liver-derived parenchymal hepatocyte growth substance (5μg/ml) obtained in step (3) above was treated with trypsin (made bySigma; 0.1 mg/ml) at 37° C. for 30 minutes. After trypsin was removed,the treated matter was added to the culture medium of the rat primaryculture hepatocytes but no growth activity was exhibited on thehepatocytes.

When the substance was treated with chymotrypsin (made by Sigma; 0.1mg/ml) at 37° C. for 30 minutes, the parenchymal hepatocyte growthactivity was also lost.

(6) Column Characteristic

1. Heparin-Sepharose

The culture medium, 1 liter, containing the parenchymal hepatocytegrowth substance and produced by genetic recombination obtained in step(3) above was dialyzed to 20 mM phosphate buffer (pH 6.5)/0.5M NaCl for16 hours and the dialysate was thoroughly equilibrated. Then thedialysate was poured through a heparin-Sepharose column (2×20 cm)equilibrated with the same buffer. After thoroughly washing with thesame buffer, elution was performed with 20 mM phosphate buffer (pH6.5)/2.0M NaCl. The parenchymal hepatocyte growth substance was found inthe non-adsorbed fraction but absent in the eluted fraction. It wasconfirmed by electrophoresis (SDS-PAGE) and by the method fordetermining the parenchymal hepatocyte growth activity of the ratprimary culture as to whether or not the substance was present. Theresults reveal that the parenchymal hepatocyte growth substance was notadsorbed to heparin-Sepharose.

2. CM Sepharose

The culture medium, 1 liter, containing the parenchymal hepatocytegrowth substance and produced by genetic recombination obtained in step(3) above was dialyzed to 20 mM phosphate buffer (pH 6.5) for 16 hours.The dialysate was thoroughly equilibrated and then poured through a CMSepharose column (2×20 cm) equilibrated with the same buffer. Afterthoroughly washing with the same buffer, elution was performed with 20mM phosphate buffer (pH 6.5)/2.0M NaCl. The parenchymal hepatocytegrowth substance was found in the non-adsorbed fraction but absent inthe eluted fraction. It was confirmed by electrophoresis (SDS-PAGE) andby the method for determining the parenchymal hepatocyte growth activityof the rat primary culture as to whether or not the substance waspresent. The results reveal that the parenchymal hepatocyte growthsubstance was not adsorbed to CM Sepharose.

3. DEAE Cellulose

The culture medium, 1 liter, containing the parenchymal hepatocytegrowth substance and produced by genetic recombination obtained in step(3) above was dialyzed to 20 mM Tris hydrochloride buffer (pH 8.0) for16 hours. The dialysate was thoroughly equilibrated and then pouredthrough a DEAE Sepharose column (2×20 cm) equilibrated with the samebuffer. After thoroughly washing with the same buffer, gradient elutionwas performed with 20 mM Tris hydrochloride buffer (pH 8.0)/2.0M NaCl.The parenchymal hepatocyte growth substance was found in a small amountin the non-adsorbed fraction and also eluted in the fraction of 0.5-0.8MNaCl. It was confirmed by electrophoresis (SDS-PAGE) and by the methodfor determining the parenchymal hepatocyte growth activity of the ratprimary culture as to whether or not the substance was present. Theresults reveal that the parenchymal hepatocyte growth substance wasalmost all adsorbed to DEAE Sepharose.

Industrial Applicability

According to the present invention, novel parenchymal hepatocyte growthsubstance originating in a human or animal liver can be obtained andthis substance possesses an activity of extracellularly proliferatingparenchymal hepatocytes. According to the present invention, there isalso provided a gene coding for the parenchymal hepatocyte growthsubstance and using the gene, the parenchymal hepatocyte growthsubstance can be mass-produced by genetic recombination technique.

Therefore, the present invention has an extreme significance inproviding a route for development of a new therapeutic agent for liverdiseases and a new agent for clinical diagnosis for hepatitis andhepatic function test.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 7                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1101 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 48..989                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 48..119                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GACCCAGCCTAGCTGAGTACTGATTCATTTTGATGTGAGTGGGAAGAATGGGGGAG56                    MetGlyGlu                                                                     ATTCGCAGCTTTGTCCTCATCACTGTTGCTCTGATTCTGGGCAAGGAG104                           IleArgSerPheValLeuIleThrValAlaLeuIleLeuGlyLysGlu                              51015                                                                         AGCTGGGTCCTCGGAGATGAGAACTGTTTGCAGGAGCAGGTCAGGCTC152                           SerTrpValLeuGlyAspGluAsnCysLeuGlnGluGlnValArgLeu                              20253035                                                                      AGGGCTCAGGTGCGCCAGCTTGAGACCCGGGTCAAACAACAACAGGTG200                           ArgAlaGlnValArgGlnLeuGluThrArgValLysGlnGlnGlnVal                              404550                                                                        GTGATTGCACAGCTCTTGCACGAGAAGGAGGTCCAGTTCCTGGATAGA248                           ValIleAlaGlnLeuLeuHisGluLysGluValGlnPheLeuAspArg                              556065                                                                        GGACAGGAGGACAGCTTCATTGACCTTGGAGGCAAGAGGCATTACGCA296                           GlyGlnGluAspSerPheIleAspLeuGlyGlyLysArgHisTyrAla                              707580                                                                        GATTGTTCAGAGATTTACAATGATGGATTTAAACATAGTGGGTTTTAC344                           AspCysSerGluIleTyrAsnAspGlyPheLysHisSerGlyPheTyr                              859095                                                                        AAAATCAAACCTCTTCAGAGTCTGGCAGAATTCTCTGTTTATTGTGAT392                           LysIleLysProLeuGlnSerLeuAlaGluPheSerValTyrCysAsp                              100105110115                                                                  ATGTCTGATGGAGGAGGATGGACTGTAATTCAGAGACGATCTGACGGC440                           MetSerAspGlyGlyGlyTrpThrValIleGlnArgArgSerAspGly                              120125130                                                                     AGTGAGAACTTTAACAGGGGTTGGAACGACTATGAAAATGGCTTTGGA488                           SerGluAsnPheAsnArgGlyTrpAsnAspTyrGluAsnGlyPheGly                              135140145                                                                     AACTTTGTCCAAAGCAATGGTGAATACTGGCTGGGTAACAAAAACATT536                           AsnPheValGlnSerAsnGlyGluTyrTrpLeuGlyAsnLysAsnIle                              150155160                                                                     AACTTGCTGACTATGCAAGGAGACTACACTTTAAAAATCGACCTGACA584                           AsnLeuLeuThrMetGlnGlyAspTyrThrLeuLysIleAspLeuThr                              165170175                                                                     GACTTTGAGAAAAACAGCCGCTTCGCACAATACGAAAAATTTAAAGTT632                           AspPheGluLysAsnSerArgPheAlaGlnTyrGluLysPheLysVal                              180185190195                                                                  GGCGATGAAAAGTCTTTTTACGAACTGAATATTGGAGAATATTCTGGC680                           GlyAspGluLysSerPheTyrGluLeuAsnIleGlyGluTyrSerGly                              200205210                                                                     ACCGCCGGAGACTCCCTGTCGGGAACATTTCACCCTGAAGTGCAGTGG728                           ThrAlaGlyAspSerLeuSerGlyThrPheHisProGluValGlnTrp                              215220225                                                                     TGGGCTAGTCACCAAACAATGAAGTTCAGCACACGGGACAGAGACAAC776                           TrpAlaSerHisGlnThrMetLysPheSerThrArgAspArgAspAsn                              230235240                                                                     GACAACTACAACGGGAACTGTGCTGAGGAGGAACAGTCTGGCTGGTGG824                           AspAsnTyrAsnGlyAsnCysAlaGluGluGluGlnSerGlyTrpTrp                              245250255                                                                     TTTAACAGGTGTCACTCTGCAAACCTGAACGGCGTGTACTACCAAGGT872                           PheAsnArgCysHisSerAlaAsnLeuAsnGlyValTyrTyrGlnGly                              260265270275                                                                  CCCTACAGAGCAGAAACCGATAATGGTGTTGTCTGGTACACCTGGCGT920                           ProTyrArgAlaGluThrAspAsnGlyValValTrpTyrThrTrpArg                              280285290                                                                     GGGTGGTGGTATTCCTTGAAATCTGTGGTTATGAAAATTAGGCCCAGT968                           GlyTrpTrpTyrSerLeuLysSerValValMetLysIleArgProSer                              295300305                                                                     GATTTTATTCCAAATATCGTTTAGTTGTCCCATTGGGATCTGCTTTCTGTG1019                       AspPheIleProAsnIleVal                                                         310                                                                           ATTCATCTTGGTTTTTAAATGTTTGAAAAAAATATACAATTCTGAATAATACACTCGTGG1079              CGATGGTGAAAAAAAAAAAAAA1101                                                    (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 314 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGlyGluIleArgSerPheValLeuIleThrValAlaLeuIleLeu                              151015                                                                        GlyLysGluSerTrpValLeuGlyAspGluAsnCysLeuGlnGluGln                              202530                                                                        ValArgLeuArgAlaGlnValArgGlnLeuGluThrArgValLysGln                              354045                                                                        GlnGlnValValIleAlaGlnLeuLeuHisGluLysGluValGlnPhe                              505560                                                                        LeuAspArgGlyGlnGluAspSerPheIleAspLeuGlyGlyLysArg                              65707580                                                                      HisTyrAlaAspCysSerGluIleTyrAsnAspGlyPheLysHisSer                              859095                                                                        GlyPheTyrLysIleLysProLeuGlnSerLeuAlaGluPheSerVal                              100105110                                                                     TyrCysAspMetSerAspGlyGlyGlyTrpThrValIleGlnArgArg                              115120125                                                                     SerAspGlySerGluAsnPheAsnArgGlyTrpAsnAspTyrGluAsn                              130135140                                                                     GlyPheGlyAsnPheValGlnSerAsnGlyGluTyrTrpLeuGlyAsn                              145150155160                                                                  LysAsnIleAsnLeuLeuThrMetGlnGlyAspTyrThrLeuLysIle                              165170175                                                                     AspLeuThrAspPheGluLysAsnSerArgPheAlaGlnTyrGluLys                              180185190                                                                     PheLysValGlyAspGluLysSerPheTyrGluLeuAsnIleGlyGlu                              195200205                                                                     TyrSerGlyThrAlaGlyAspSerLeuSerGlyThrPheHisProGlu                              210215220                                                                     ValGlnTrpTrpAlaSerHisGlnThrMetLysPheSerThrArgAsp                              225230235240                                                                  ArgAspAsnAspAsnTyrAsnGlyAsnCysAlaGluGluGluGlnSer                              245250255                                                                     GlyTrpTrpPheAsnArgCysHisSerAlaAsnLeuAsnGlyValTyr                              260265270                                                                     TyrGlnGlyProTyrArgAlaGluThrAspAsnGlyValValTrpTyr                              275280285                                                                     ThrTrpArgGlyTrpTrpTyrSerLeuLysSerValValMetLysIle                              290295300                                                                     ArgProSerAspPheIleProAsnIleVal                                                305310                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1093 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 51..986                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 51..116                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TGAGCTGGGTCTCTGACTCACTTCTGACTTTAGTTTTTTCAAGGGGGAACATGGCA56                    MetAla                                                                        1                                                                             AAGGTGTTCAGTTTCATCCTTGTTACCACCGCTCTGATAATGGGCAGG104                           LysValPheSerPheIleLeuValThrThrAlaLeuIleMetGlyArg                              51015                                                                         GAAATTTCGGCGCTCGAGGACTGTGCCCAGGAGCAGATGCGGCTCAGA152                           GluIleSerAlaLeuGluAspCysAlaGlnGluGlnMetArgLeuArg                              202530                                                                        GCCCAGGTGCGCCTGCTTGAGACCCGGGTCAAACAGCAACAGGTCAAG200                           AlaGlnValArgLeuLeuGluThrArgValLysGlnGlnGlnValLys                              35404550                                                                      ATCAAGCAGCTTTTGCAGGAGAATGAAGTCCAGTTCCTTGATAAAGGA248                           IleLysGlnLeuLeuGlnGluAsnGluValGlnPheLeuAspLysGly                              556065                                                                        GATGAGAATACTGTCGTTGATCTTGGAAGCAAGAGGCAGTATGCAGAT296                           AspGluAsnThrValValAspLeuGlySerLysArgGlnTyrAlaAsp                              707580                                                                        TGTTCAGAGATTTTCAATGATGGGTATAAGCTCAGTGGATTTTACAAA344                           CysSerGluIlePheAsnAspGlyTyrLysLeuSerGlyPheTyrLys                              859095                                                                        ATCAAACCTCTCCAGAGCCCAGCAGAATTTTCTGTTTATTGTGACATG392                           IleLysProLeuGlnSerProAlaGluPheSerValTyrCysAspMet                              100105110                                                                     TCCGATGGAGGAGGATGGACTGTAATTCAGAGACGATCTGATGGCAGT440                           SerAspGlyGlyGlyTrpThrValIleGlnArgArgSerAspGlySer                              115120125130                                                                  GAAAACTTTAACAGAGGATGGAAAGACTATGAAAATGGCTTTGGAAAT488                           GluAsnPheAsnArgGlyTrpLysAspTyrGluAsnGlyPheGlyAsn                              135140145                                                                     TTTGTCCAAAAACATGGTGAATATTGGCTGGGCAATAAAAATCTTCAC536                           PheValGlnLysHisGlyGluTyrTrpLeuGlyAsnLysAsnLeuHis                              150155160                                                                     TTCTTGACCACTCAAGAAGACTACACTTTAAAAATCGACCTTGCAGAT584                           PheLeuThrThrGlnGluAspTyrThrLeuLysIleAspLeuAlaAsp                              165170175                                                                     TTTGAAAAAAATAGCCGTTATGCACAATATAAGAATTTCAAAGTTGGA632                           PheGluLysAsnSerArgTyrAlaGlnTyrLysAsnPheLysValGly                              180185190                                                                     GATGAAAAGAATTTCTACGAGTTGAATATTGGGGAATATTCTGGAACA680                           AspGluLysAsnPheTyrGluLeuAsnIleGlyGluTyrSerGlyThr                              195200205210                                                                  GCTGGAGATTCCCTTGCGGGGAATTTTCATCCTGAGGTGCAGTGGTGG728                           AlaGlyAspSerLeuAlaGlyAsnPheHisProGluValGlnTrpTrp                              215220225                                                                     GCTAGTCACCAAAGAATGAAATTCAGCACGTGGGACAGAGATCATGAC776                           AlaSerHisGlnArgMetLysPheSerThrTrpAspArgAspHisAsp                              230235240                                                                     AACTATGAAGGGAACTGCGCAGAAGAAGATCAGTCTGGCTGGTGGTTT824                           AsnTyrGluGlyAsnCysAlaGluGluAspGlnSerGlyTrpTrpPhe                              245250255                                                                     AACAGGTGTCACTCTGCAAACCTGAATGGTGTATACTACAGCGGCCCC872                           AsnArgCysHisSerAlaAsnLeuAsnGlyValTyrTyrSerGlyPro                              260265270                                                                     TACACGGCTAAAACAGACAATGGGATTGTCTGGTACACCTGGCATGGG920                           TyrThrAlaLysThrAspAsnGlyIleValTrpTyrThrTrpHisGly                              275280285290                                                                  TGGTGGTATTCTCTGAAATCTGTGGTTATGAAAATTAGGCCAAATGAT968                           TrpTrpTyrSerLeuLysSerValValMetLysIleArgProAsnAsp                              295300305                                                                     TTTATTCCAAATGTAATTTAATTGCTGCTGTTGGGCTTTCGTTTCTGC1016                          PheIleProAsnValIle                                                            310                                                                           AATTCAGCTTTGTTTAAAGTGATTTGAAAAATACTCATTCTGAACATATCCAGCGCAATC1076              ATGATAACTGTTGTGAG1093                                                         (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 312 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetAlaLysValPheSerPheIleLeuValThrThrAlaLeuIleMet                              151015                                                                        GlyArgGluIleSerAlaLeuGluAspCysAlaGlnGluGlnMetArg                              202530                                                                        LeuArgAlaGlnValArgLeuLeuGluThrArgValLysGlnGlnGln                              354045                                                                        ValLysIleLysGlnLeuLeuGlnGluAsnGluValGlnPheLeuAsp                              505560                                                                        LysGlyAspGluAsnThrValValAspLeuGlySerLysArgGlnTyr                              65707580                                                                      AlaAspCysSerGluIlePheAsnAspGlyTyrLysLeuSerGlyPhe                              859095                                                                        TyrLysIleLysProLeuGlnSerProAlaGluPheSerValTyrCys                              100105110                                                                     AspMetSerAspGlyGlyGlyTrpThrValIleGlnArgArgSerAsp                              115120125                                                                     GlySerGluAsnPheAsnArgGlyTrpLysAspTyrGluAsnGlyPhe                              130135140                                                                     GlyAsnPheValGlnLysHisGlyGluTyrTrpLeuGlyAsnLysAsn                              145150155160                                                                  LeuHisPheLeuThrThrGlnGluAspTyrThrLeuLysIleAspLeu                              165170175                                                                     AlaAspPheGluLysAsnSerArgTyrAlaGlnTyrLysAsnPheLys                              180185190                                                                     ValGlyAspGluLysAsnPheTyrGluLeuAsnIleGlyGluTyrSer                              195200205                                                                     GlyThrAlaGlyAspSerLeuAlaGlyAsnPheHisProGluValGln                              210215220                                                                     TrpTrpAlaSerHisGlnArgMetLysPheSerThrTrpAspArgAsp                              225230235240                                                                  HisAspAsnTyrGluGlyAsnCysAlaGluGluAspGlnSerGlyTrp                              245250255                                                                     TrpPheAsnArgCysHisSerAlaAsnLeuAsnGlyValTyrTyrSer                              260265270                                                                     GlyProTyrThrAlaLysThrAspAsnGlyIleValTrpTyrThrTrp                              275280285                                                                     HisGlyTrpTrpTyrSerLeuLysSerValValMetLysIleArgPro                              290295300                                                                     AsnAspPheIleProAsnValIle                                                      305310                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       LeuGluAspCysAlaGlnGluGlnMetArgLeuArgAlaGlnValArg                              151015                                                                        LeuLeuGluThrArgValLysGlnGlnGlnValLysIleLys                                    202530                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AspGluAsnCysLeuGlnGluGlnValArgLeuArgAlaGlnValArg                              151015                                                                        GlnLeuGluThrArgValLysGlnGlnGlnValValIleAla                                    202530                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TGCCGTCAGATCGTCTCTGAATTACAGTCCATCCTCCTCC40                                    __________________________________________________________________________

We claim:
 1. An isolated and purified parenchymal hepatocyte growthfactor substance which is naturally produced in a non-nalion liver andhas the following physicochemical properties and physiologicalactivities:(1) an estimated molecular weight according to nonreductiveSDS-PAGE of about 63,000 to about 69,000, an estimated molecular weightaccording to reductive SDS-PAGE of about 32,000 to about 36,000 and anestimated molecular weight according to gel filtration of about 60 toabout 70 Kd; (2) an activity of effecting the growth of parenchymalhepatocytes; (3) said activity of effecting the growth of parenchymalhepatocytes being lost by a heat treatment at 95° C. for 5 minutes, atreatment with trypsin or a treatment with chymotrypsin; (4) adsorptionto DEAE Sepharose resin at pH 8.0 in 20 mM Tris hydrochloride buffer;and (5) non-adsorption to heparin.
 2. A parenchymal hepatocyte growthfactor to claim 1, having an N-terminal amino acid sequence of SEQ IDNO:5.
 3. A parenchymal hepatocyte growth factor according to claim 1,having an N-terminal amino acid sequence of SEQ ID NO:6.
 4. Aparenchymal hepatocyte growth factor according to claim 2, wherein saidfactor has an amino acid sequence from 23 leucine to 312 isoleucine inthe amino acid sequence of SEQ ID NO:4.
 5. A parenchymal hepatocytegrowth factor according to claim 3, wherein said factor has an aminoacid sequence from 25 aspartic acid to 314 valine in the amino acidsequence of SEQ ID NO:2.
 6. An isolated DNA coding for a parenchymalhepatocyte growth factor having an amino acid sequence from 23 leucineto 312 isoleucine in the amino acid sequence of SEQ ID NO:4.
 7. Anisolated DNA coding for a parenchymal hepatocyte growth substance havingan amino acid sequence from 25 aspartic acid to 314 valine in the aminoacid sequence of SEQ ID NO:2.
 8. A process for producing a parenchymalhepatocyte growth factor according to claim 1, which comprisestransforming a host cell with an expression vector comprising a DNAcoding for a parenchymal hepatocyte growth factor having an amino acidsequence from 23 leucine to 312 isoleucine in the amino acid sequence ofSEQ ID NO:4, culturing the resulting transformant and recovering theparenchymal hepatocyte growth factor from the culture medium.
 9. Aprocess for producing a parenchymal hepatocyte growth factor accordingto claim 1, which comprises transforming a host cell with an expressionvector comprising a DNA coding for a parenchymal hepatocyte growthfactor having an amino acid sequence from 25 aspartic acid to 314 valinein the amino acid sequence of SEQ ID NO:2, culturing the resultingtransformant and recovering the parenchymal hepatocyte growth factorfrom the culture medium.
 10. A process for isolating a parenchymalhepatocyte growth factor according to claim 1, comprising:contacting anantibody to a peptide having the sequence of amino acids 180 to 201 ofSEQ ID NO:2 with a homogenate of a mammalian liver after a partialhepatectomy of said liver to form an antibody-parenchymal hepatocytegrowth factor complex; and isolating said parenchymal hepatocyte growthfactor from said complex.
 11. The process according to claim 10, whereinsaid antibody is bound to a column.